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A series of Special Issues of Neuropharmacology celebrates the 40th anniversary of a seminal review on excitatory amino acid (EAA) receptors by two pioneers of the field – Dick Evans and Jeff Watkins. Brought together in the Department of Pharmacology at the University of Bristol in the 1970s, they forged a partnership that, through the synthetic chemistry prowess of Jeff Watkins, which provided novel agonists and antagonists for EAA receptors for Dick Evans's deft experimental studies, generated enormous insight into the multitude of actions of EAAs in the nervous system. Among many achievements from this time was not just the naming of the N-methyl- d -aspartate (NMDA) receptor, but also the demonstration of its antagonism by magnesium ions. Here, Dick and Jeff reflect upon those early halcyon days of EAA research, which, as these six 1 Special Issues of Neuropharmacology demonstrate, is very much alive and kicking. Bruno G. Frenguelli, Editor-in-Chief, Neuropharmacology.

In 1981 Jeff Watkins and Dick Evans wrote what was to become a seminal review on excitatory amino acids (EAAs) and their receptors (Watkins and Evans, 1981). Bringing together various lines of evidence dating back over several decades on: the distribution in the nervous system of putative amino acid neurotransmitters; enzymes involved in their production and metabolism; the uptake and release of amino acids; binding of EAAs to membranes; the pharmacological action of endogenous excitatory amino acids and their synthetic analogues, and notably the actions of antagonists for the excitations caused by both nerve stimulation and exogenous agonists, often using pharmacological tools developed by Jeff and his colleagues, they provided a compelling account for EAAs, especially l -glutamate, as a bona fide neurotransmitter in the nervous system. The rest, as they say, is history, but far from being consigned to history, EAA research is in rude health well into the 21st Century as this series of Special Issues of Neuropharmacology exemplifies. With EAAs and their receptors flourishing across a wide range of disciplines and clinical conditions, we enter into a dialogue with two of the most prominent and influential figures in the early days of EAA research: Jeff Watkins and Dick Evans.

SummaryNiemann–Pick type C (NP-C) disease is a rare neurodegenerative lysosomal storage disorder. It is highly heterogeneous, and there is limited awareness of a substantial subgroup that has an attenuated adolescent/adult-onset disease. In these patients psychiatric features, often a psychosis, may dominate the initial impression, although often there is an associated ataxia and cognitive impairment. Typically, patients experience a substantial diagnostic delay. In this review we highlight the importance of early recognition and discuss the pathophysiology, neuropsychiatric presentation and recent changes in the investigation and work-up of these patients, and treatment options.

The potential anticancer agent fostriecin (FOS) is a potent inhibitor of the protein Ser/Thr phosphatases PP2A and PP4 and a weaker inhibitor of PP1. Random mutagenesis and automated screening in yeast identified residues in human PP2Acalpha important for inhibitory FOS binding. A C269S substitution in the predicted beta12-beta13 loop decreased the FOS sensitivity of intact cells and increased the IC(50) of PP2Acalpha by 10-fold in vitro. Changing PP2Acalpha Cys-269 to phenylalanine, the equivalent residue in PP1, and the Y267G and G270D substitutions caused a similar effect. The results provide information relevant to the design of novel protein Ser/Thr phosphatase inhibitory drugs.

α2-Adrenoceptor agonists have a spinal site of analgesic action. In the current study the synaptic depressant actions of xylazine, detomidine, romifidine and dexmedetomidine have been compared on segmental reflexes containing NMDA receptor-mediated components in the neonatal rat hemisected spinal cord preparation in vitro. Reflexes were evoked in the ventral root following either supramaximal electrical stimulation of the corresponding ipsilateral lumbar dorsal root to evoke the high intensity excitatory postsynaptic potential (e.p.s.p.) involving all primary afferent fibres, or low intensity stimulation to evoke the solely A fibre-mediated low intensity e.p.s.p. The high intensity e.p.s.p. contains a greater NMDA receptor-mediated component. Xylazine, romifidine, detomidine and dexmedetomidine all depressed both the high intensity e.p.s.p. and the low intensity e.p.s.p. giving respective EC50 values of 0.91±0.2 μM (n=12), 23.4±3 nM (n=12), 37.7±7 nM (n=8) and 0.84±0.1 nM (n=4) for depression of the high intensity e.p.s.p. and 0.76±0.1 μM (n=12), 22.0±3 nM (n=12), 24.9±6 nM (n=4) and 2.7±0.6 nM (n=4) for depression of the low intensity e.p.s.p., respectively. Unlike the other three drugs, the two values for dexmedetomidine, showing a greater selectivity for the high intensity e.p.s.p., are significantly different. Each of these depressant actions was reversed by the selective α2-adrenoceptor antagonist atipamezole (1 μM). In contrast to previous reports of the actions of α2-adrenoceptor agonists on the in vitro spinal cord preparation, at concentrations ten fold higher than the above EC50 values xylazine, romifidine, detomidine and dexmedetomidine depressed the initial population spike of motoneurons (MSR). This depression was not reversed by atipamezole. Comparison of the rank order of the present EC50 values for depression of the high intensity e.p.s.p. with potency ratios from in vivo analgesic tests in previous studies show a close correlation between the present in vitro tests and analgesic potency. There is no correlation between the present data and previously obtained affinities of the agonists at non-adrenergic imidazoline binding sites. The current findings therefore suggest that xylazine, romifidine, detomidine and dexmedetomidine are exerting their central analgesic actions at the spinal level principally through α-2-adrenoceptors. All four agonists showed the same profile of selective depression of the NMDA receptor-mediated component of reflexes similar to that reported previously for clonidine. However dexmedetomidine, unlike the other ligands, selectively depressed the high intensity e.p.s.p.

The present study aimed to compare the actions of the selective κ opioid receptor agonist enadoline (CI-977) with morphine in order to see if there is a heterogeneity of opioid receptors between spinal reflex pathways. High (C- and A-fibre evoked activity) and low (A-fibres only) intensity electrical stimulation of dorsal roots in the neonatal rat hemisected spinal cord preparation in vitro was used to distinguish between synaptic activity measured in the corresponding ventral root. Enadoline selectively depressed the high intensity-evoked EPSP with an EC 50 of 7.6 nM ( n =7), contrasting with our previous finding in this preparation that morphine is an equipotent depressant of A- and C-fibre-mediated synaptic responses. The depressant effects of enadoline and morphine were reversed by naloxone giving apparent K d values of 14±3 nM ( n =4) for enadoline-induced and 4.2±1 nM ( n =4) for morphine-induced depression. These data suggest that activation of κ opioid receptors has a selective depressant action on C-fibre-mediated synaptic activity. Such a functional difference mediated at a subclass of opioid receptors has not been previously observed in an in vitro spinal preparation.

A 29-year-old Caucasian woman presented to hospital with a 2-day history of diarrhoea, anorexia and rigors. Investigations showed abnormal liver function tests, hyponatremia, hypoalbuminaemia and lymphopenia. The initial chest radiograph was normal. A bone marrow trephine biopsy showed non-caseating granulomata and she subsequently developed miliary shadowing on the chest radiograph. A transjugular liver biopsy confirmed the presence of acid-alcohol fast bacilli. Despite starting triple therapy for miliary tuberculosis she remained febrile and developed massive hepatosplenomegaly, jaundice and pancytopenia. Standard triple therapy was substituted with ethambutol, streptomycin and oral prednisolone and the patient made a dramatic recovery. The clinical symptoms of miliary tuberculosis are frequently non-specific and the onset of the illness is often insidious. The liver is involved in almost all patients with miliary tuberculosis, but massive hepatosplenomegaly and jaundice are rare. Standard triple-therapy should be discontinued when there is significant liver dysfunction, and corticosteroids should be considered for patients with miliary tuberculosis who fail to respond to conventional therapy.

Mutations in the Schizosaccharomyces pombe rec14 gene reduce meiotic recombination by as much as a factor of 1000 in the three intervals tested on chromosomes I and III. A DNA clone complementing the rec14 mutation was shown by genetic and physical analysis to contain the rec14 gene, which was functional in plasmid-borne inserts as small as 1.4 kb. The rec14 gene contains two exons separated by a 53-bp intron, which was confirmed by analysis of rec14 transcripts. The spliced transcript encodes a protein product of 302 amino acids, which contains six WD repeat motifs found in the G-beta transducin family of proteins and other proteins, including the Saccharomyces cerevisiae Ski8 (Rec103) protein. Although the rec14 transcripts were present in mitotically dividing cells, rec14 mutations had no detectable effect on mitotic recombination. The pattern of expression of rec14 differs from that of previously analyzed S. pombe rec genes. Based upon mutant phenotypes and amino acid sequence similarities, we propose that S. pombe Rec14 is a functional homologue of S. cerevisiae Rec103.

1. Whole cell synaptic currents were recorded under voltage clamp from a total of 54 ventral horn neurones held near to their resting potential by the patch clamp technique in immature rat spinal cord preparations in vitro. Twenty eight neurones were identified, by antidromic invasion from ventral roots, as motoneurones. Excitatory postsynaptic currents (e.p.s.cs) of peak amplitude -480 pA +/- 66 s.e. mean and -829 +/- 124 pA were evoked respectively from the unidentified ventral horn neurones and the motoneurones in response to maximal activation of the segmental dorsal root. 2. The e.p.s.cs were depressed reversibly by the metabotropic glutamate agonists 1S3S-1-aminocyclopentane-1,3-dicarboxylate (1S3S-ACPD) (EC50 17.1 microM +/- 0.3 s.e. mean, n = 14) and L-2-amino-4-phosphonobutanoate (L-AP4) (EC50 = 2.19 +/- 0.19 microM, n = 15). Since both agonists independently produced more than 90% depression it is likely that the receptors that mediate their effects are present on the same presynaptic terminals. 3. When the Mg2+ concentration was raised from 0.75 mM to 2.75 mM together with the addition of 50 microM D-2-amino-5-phosphonopentanoate (AP5), a treatment which would increase the proportion of monosynaptic component in the e.p.s.c. the concentration-effect plots for both 1S3S-ACPD (EC50 1.95 +/- 0.4 microM, n = 8) and L-AP4 (EC50 0.55 +/- 0.20 microM, n = 7) were shifted to the left, suggesting that monosynaptic e.p.cs of primary afferents to ventral horn neurones are more susceptible to L-AP4 and 1S3S-ACPD than are other synapses in polysynaptic pathways. 4. lS3S-ACPD (20 and 50 microM) also caused mean sustained inward currents of 95 +/- 31 pA (n = 6) and248 +/- 49 pA (n = 10) respectively. In the combined presence of AP5 (50 microM) and Mg2+ (2.75 mM) themean response to 50 microM lS3S-ACPD was reduced to 106+/- 18 pA (n = 4). In the presence of tetrodotoxin(1 microM) the corresponding value was 48 +/- 6 pA (n = 4). Similar sustained inward currents produced by N-methyl-D-aspartate (NMDA) were almost abolished to10 pA in the presence of AP5 and 2.75 mMMg2+. In the presence of tetrodotoxin the maximum inward current produced by NMDA was undiminished. Thus a large component of the excitatory action of lS3S-ACPD was mediated at non-NMDA receptors both directly at the patch-clamped neurones and indirectly by synaptic relay.

The goal of these investigations was to study the role of tachykinin NK2 receptors in neonatal spinal cords using the selective NK2 receptor agonist [β-Ala8]neurokinin A-(4–10) and the new NK2 receptor antagonist GR 94800. Experiments were performed with superfused hemisected rat and gerbil spinal cords. Dorsal roots were electrically stimulated and the synaptically elicited responses and the DC-potentials were recorded extracellularly from the corresponding ventral roots. [β-Ala8]neurokinin A-(4–10) depolarized ventral roots (0.01–10 μM) and increased their spontaneous activity in a concentration-dependent manner. These effects of [β-Ala8]neurokinin A-(4–10) were reduced by GR 94800. The action of GR 94800 was selective because the depolarizing effects of similar magnitude evoked by the NK1 receptor agonist [Sar9, Met(O2)11]substance P were not affected by GR 94800. The pA2 values of GR 94800 amounted to 6.0±0.4 in the rat and 5.4±0.3 in the gerbil. The NK2 receptor agonist was more potent in the rat than in the gerbil. The estimated EC50 (means ± S.E.M.) was found to be 3.9 + 6.0/ − 1.3 μM in the rat and 2.4 + 2.9/ − 1.3 μM in the gerbil spinal cord. The NK2 receptor agonist [β-Ala8]neurokinin A-(4–10) potentiated the monosynaptic reflex evoked by dorsal root stimulation. The potentiation manifested itself as an increase in the amplitude of the early component of the response. The receptor type mediating this effect could not be elucidated. The potentiation ranged between 30 ± 27 and 110 ± 36% (0.3 and 10 μM), respectively. Tetrodotoxin reduced the depolarizing action elicited by the NK2 receptor agonist, suggesting that part of the [β-Ala8]neurokinin A-(4–10) effect is probably located presynaptically. In contrast to the NK1 receptor antagonist (±)-CP-96,345, the NK2 receptor anatgonist GR 94800 showed no species differences. In conclusion, NK2 receptors appear to have a physiological role in regulating spinal neuronal activity.

The potencies of two GABAB receptor antagonists P-[3-aminopropyl]- P-diethoxymethyl-phosphinic acid (CGP35348) and the novel compound 3-N[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-P- benzyl-phosphinic acid (CGP55845) have been compared in an in vitro spinal cord preparation. They have been tested as antagonists of baclofen-induced depression of EPSCs of patch-clamped dorsal horn neurons following electrical stimulation of dorsal roots. Mean EC50 values for the depressant action of baclofen were increased by 50- and 140-fold respectively in the presence of CGP35348 (200 microM) (n = 5) and CGP55845 (100 nM) (n = 4). This potency of CGP55845 is > 1000-fold higher than that reported previously for other GABAB receptor antagonists.

1. The effect of the myorelaxant drugs baclofen, diazepam and tizanidine have been compared on in vitro preparations of baby rat spinal cord and adult rat superior cervical ganglion. 2. Dorsal root-elicited long duration (time to half decay 9.71 +/- 0.29 s.e. mean, n = 31) ipsilateral ventral root reflexes (DR-VRP), measured as integrated area, of immature rat spinal cord preparations were abolished by RS-2-amino-5-phosphonopentanoate (AP5) (EC50 8.13 +/- 0.92 microM, n = 3). The initial short latency component of DR-VRP was resistant to AP5. 3. Baclofen abolished both components of the DR-VRP. Respective EC50 values for the AP5-insensitive and AP5-sensitive components were 237 +/- 68 nM (n +/- 7) and 57 +/- 10 nM (n = 7). These effects of baclofen were reversed by the GABAB antagonist, CGP35348. The apparent Kd values (16.7 +/- 6.4 microM, n = 3 and 14.3 +/- 3.9 microM, n = 6 respectively) for this reversal were not significantly different. 4. Tizanidine, clonidine and diazepam had no effect on the AP5-insensitive component of the DR-VRP. 5. The AP5-sensitive long duration component of the DR-VRP was depressed to respective maximal levels of 23.2 +/- 1.4% (n = 7), 18.8 +/- 3.8% (n = 4) and 47.6 +/- 1.6% (n = 5) of control (100%) levels by tizanidine (EC50 135 +/- 33 nM), clonidine (EC50 26.0 +/- 2.2 nM) and diazepam (EC25 114 +/- 12 nM, n = 4). The depressant effects of tizanidine and clonidine were reversed by idazoxan (1 microM). Flumazenil (I microM) failed to reverse the depressant effect of tizanidine. The depressant effect of diazepam was reversed by flumazenil (1 microM) but not by idazoxan (1 microM). Naloxone 1 M did not reverse the effects of either tizanidine or diazepam.6. In the presence of tetrodotoxin (0.1 SAM) which abolished synaptic activity, clonidine, tizanidine or diazepam (10, 100 and 101JM respectively) produced no significant antagonism of NMDA-induced depolarizations recorded from ventral roots.7. Control (100%) synaptic responses of rat superior cervical ganglion preparations were depressed respectively to near maximal levels of 60.0 +/- 5.2% (n = 4) and 60.7 +/- 5.6% (n = 5) by clonidine (0.5 JAM,EC25 15.3 +/- 3.0 nM) and tizanidine (1 JAM, EC25 227 +/- 83 nM). These depressant effects were reversed by idazoxan (1 AM). Baclofen (EC25 28.7 +/- 10.0, n = 3) depressed the postganglionic response to a maximum level of 71.8 + 2.4% (n = 4) control at a concentration of 100 microM. The latter depressant action was reversed by the GABAB receptor antagonist, CGP35348 (1 mM). Diazepam (1 microM) had no significant effect on ganglionic transmission.8. It is concluded that the activation of benzodiazepine or M2-noradrenaline receptors can modulate NMDA receptor-mediated excitatory synaptic pathways whereas synaptic excitation from primary afferent terminals, mediated by non-NMDA receptors, appears to lack the propensity for this type of modulation. The results show also that the isolated spinal preparation can be used to identify central myorelaxant actions that are mediated through functional benzodiazepine or X2-noradrenaline receptors.

6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM) depressed dorsal root-evoked ventral and dorsal root potentials of the in vitro immature rat spinal cord to 26.3 +/- 5.2 S.E.M. and 40.8 +/- 2.7% of control values respectively. These depressant effects of CNQX were partially reversed by D-serine (EC50 values 39.7 microM +/- 8.7 S.E.M. N = 6 and 34.9 +/- 12.5 microM, N = 5 for ventral root potential and dorsal root potential respectively). Under our experimental conditions, which included the presence of Mg2+ (0.75 mM) in the bathing medium, no measurable potentiation of these synaptic reflexes by D-serine was recorded in the absence of CNQX. These data indicate that CNQX, in addition to its depressant effect at non-NMDA receptors, depresses an NMDA receptor-mediated component of segmental transmission through its action at the glycine site of the NMDA receptor complex.

1. The C elevation of the compound action potential (CAP) was recorded with suction electrodes from dorsal roots of rats at 25 degrees C and toads (Bufo bufo) at 10 degrees C. The C fibre CAP had a conduction velocity of 0.5 +/- 0.07 SE M per sec (N = 10) and 0.25 +/- 0.04 M per sec (N = 8) in the rat and toad nerves respectively. 2. The depressant effect of applied drugs on the amplitude of the C fibres CAP was measured. Nerves from both species had similar sensitivities to GABA. EC50 5.0 microM +/- 0.5 SEM (N = 3) and 5.5 microM +/- 1.4 (N = 3) for the rat and toad respectively. Maximum depressant effects of GABA produced in rat and toad nerves were 35% +/- 5 SEM and 17% +/- 2.5 respectively. 3. In five out of ten of the rat nerves tested kainate had a clear depressant effect (maximum 36% +/- 4.3 SEM, EC50 6.8 microM +/- 0.9 SEM, N = 3) on the C fibre CAP. Kainate, at concentrations from 100 to 500 microM, had no effect on seven toad nerves. 4. Toad nerves were about 100 times less sensitive, than rat nerves, to capsaicin (ED50 values 430 microM +/- 190 SEM and 0.7 microM +/- 0.2 respectively, N = 4). 5. The similar sensitivity of nerves in both species to GABA and differing sensitivities to kainate and capsaicin suggests that amphibian C fibres specifically lack sensitivity to capsaicin and kainate.

Electrical stimulus intensity, capsaicin, excitatory amino acid antagonists and the substance P antagonist, spantide, have been used to investigate the roles of primary afferent C fibres and excitatory amino acid receptors in the generation of long duration (half time 9.1 s +/- 1.1 S.E.M., N = 24) contralateral reflexes recorded in ventral roots of immature rat spinal cords in vitro. The relationship between C fibre compound action potentials recorded in the dorsal root and duration of the dorsal root-evoked contralateral ventral root potential appeared to be coincidental rather than causal. Dorsal root-evoked contralateral ventral root potentials of greater than 2 s in duration could not be evoked in mature mouse spinal preparations. Application of capsaicin (1 microM for 15-120 min) produced a long lasting increase in spontaneous activity of ventral roots as well as blockade of C fibre conduction in dorsal roots. The dorsal root potential evoked following stimulation of adjacent dorsal roots at intensities insufficient for activation of C fibres was depressed by capsaicin. Dorsal root-evoked contralateral ventral root potentials were abolished by kynurenate (EC50 56 +/- 13 microM, N = 3) and depressed to 38.2 +/- 6.9% S.E.M. (N = 7) of pre-drug levels by the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonopentanoate (20 microM) or to 51.8 +/- 9.0% (N = 7) by the substance P analogue spantide (33 microM). Spantide consistently antagonised substance P-induced, but not capsaicin-induced, depolarizations recorded in ventral roots (+-)-2-Amino-5-phosphonopentanoic acid (10-50 microM) depressed both substance P- and capsaicin-induced depolarizations. The depressant effect of spantide, unlike that of (+/-)-2-amino-5-phosphonopentanoic acid, was associated with a long lasting excitatory action. In the presence of tetrodotoxin (0.1 microM), spantide (33 microM) failed to antagonize substance P-induced depolarizations. It is suggested that long duration of the dorsal root-evoked contralateral ventral root potential is a consequence of the activation of the N-methyl-D-aspartate receptor operated ion channels by excitatory amino acid transmitters.

An hydrophobic interaction chromatography step was developed for the large-scale production of an Fc-fusion biologic. Two abundant product-related impurities were separated from the active monomer using a Butyl resin and a simple step-wash and step-elution strategy. Capacity and resolution of the HIC step was optimal when sodium sulfate was employed as the lyotropic salt and pore size of the Butyl resin was 750 A. Factorial analysis identified critical parameters for the Butyl chromatography and an operating window capable of delivering high product quality and yield over a broad column loading range.

The novel tachykinin receptor antagonist CGP49823 ((2R,4S)-2-benzyl-1-(3,5-dimethylbenzoyl)-4-(quinolin-4-y lmethylamino)piperidine) has been compared with three other selective non-peptide tachykinin NK1 receptor antagonists. The drugs were tested as antagonists of the depolarization of spinal motoneurones induced by bath application of the selective tachykinin NK1 receptor agonist septide-(6-11) (300 nM) for 120 s at 15 min intervals. The antagonists were bath applied and the depolarizations were recorded from lumbar ventral roots of 7 to 12 day old rat and gerbil hemisected spinal cords in vitro. The gerbil preparation is considered to model the human species variant of the tachykinin NK1 receptor. With the exception of SR140333 ((S)-1-[2-[3-(3,4-dichlorophenyl)-1-[[3-(1-methylethoxy)phenyl]ace tyl]-3-piperidinyl]ethyl]-4-phenyl-1-azoniabicyclo[2.2.2]octane chloride), the antagonists were approximately thirty-fold more potent on gerbil preparations. The respective mean IC50 values from gerbil preparations produced by CP96345 ((2S-cis)-2-(diphenylmethyl)-N-[(2-methoxyphenyl)methyl]-1-azabicy clo[2.2.2]octan-3-amine), CGP49823, SR140333 and CP99994 ((2S-cis)-N-[(2-methoxyphenyl)methyl]-2-phenyl-3-piperidinamine) were, in microM +/- S.E. (n) 0.10 +/- 0.02 (6), 0.22 +/- 0.03 (6), 0.30 +/- 0.10 (5) and 0.38 +/- 0.02 (5) and the corresponding values from the rat preparations were 3.7 +/- 0.4 (5), 7.8 + 1.3 (5), 1.06 +/- 0.16 (6) and 10.5 +/- 2.2 (7). Dominance of tachykinin NK1 receptor activity in the measured responses was confirmed by low potency of the tachykinin NK2-selective antagonist SR48968 ((S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl)butyl] benzamide) which yielded an IC50 value of 12.0 +/- 2.8 (5) on gerbil preparations and produced less than 50% depression of septide-induced depolarization of rat motoneurones at the highest concentration (100 microM) tested.

Recordings of whole-cell synaptic current responses elicited by electrical stimulation of dorsal roots were made from motoneurons, identified by antidromic invasion, in isolated spinal cord preparations from five- to eight-day-old Wistar rats. Supramaximal electrical stimulation of the dorsal root evoked complex excitatory postsynaptic currents with mean latencies (±S.E.M.) of 6.1±0.26 ms, peak amplitude of −650±47 pA and duration of 4.30±0.46 s ( n =34). All phases of excitatory postsynaptic currents were potentiated to approximately 20% above control levels in the presence of the metabotropic glutamate receptor antagonists S -2-amino-2-methyl-4-phosphonobutanoate (MAP4; 200 μ M; n =15) and 2 S ,1′ S ,2′ S -2-methyl-2-(carboxycyclopropyl)glycine (MCCG; 200 μ M; n =9). A similar level of potentiation was produced by the GABA B receptor antagonist 3- N [1-( S )-(3,4-dichlorophenyl)ethyl]amino-2-( S )-hydroxypropyl- P -benzyl-phosphinic acid (CGP55845; 200 nM; n =5). MAP4 (200 μ M) produced a six-fold rightward shift in the concentration–effect plot for the depressant action of the metabotropic glutamate receptor agonist S -2-amino-4-phosphonobutanoate ( L -AP4), whereas CGP55845 produced no significant change in the potency of l -AP4. MAP4 did not antagonize the depressant actions of baclofen ( n =8), 1 S ,3 S -1-aminocyclopentane-1,3-dicarboxylate ( n =4) or 2 S , 1′ S , 2′ S -2-(carboxycyclopropyle) glycine ( n =4). The metabotropic glutamate receptor antagonists produced no change in the holding current of any of the neurons, indicating that they had no significant postsynaptic excitatory actions. These results are the first to indicate a possible physiological role for metabotropic glutamate receptors in the spinal cord. Like GABA B receptors, they control glutamatergic synaptic transmission in the segmental spinal pathway to motoneurons. This is likely to be a presynaptic control mechanism.

The patch-clamp technique has been used to record synaptic responses, elicited by electrical stimulation of dorsal roots, in 28 single motoneurones of in vitro spinal cord preparations from neonate (P5 to P8) rats. The effects of (RS)-α-methyl-4-phosphonophenylglycine (MPPG) (200 μM), a potent antagonist at l-2-amino-4-phosphonobutanoate (AP4)-sensitive receptors, and (RS)-α-methyl-4-carboxyphenylglycine (MCPG) (500 μM), which is a less selective antagonist of mGluRs, were tested on EPSCs alone and as antagonists of AP4-induced depression of EPSCs. The ec50 for depression of EPSCs by AP4 (1.16 ± 0.12 μM, n = 8) was increased to 18.9 ± 0.7 μM (n = 6) by MPPG. MCPG (500 μM) had no significant effect on the depressant potency of AP4. Under control conditions, EPSCs had mean peak amplitudes of 983 pA ± 64 SEM and mean charge transferred of 306 ± 37 pC (n = 28). These values were increased significantly (p < 0.05) to 1168 ± 68 pA and 363 ± 39 pC by MPPG (n = 6), and 1150 ± 54 pA and 358 ± 33 pC (n = 6) by MCPG. There was no significant difference between the enhancement of the initial peak of the EPSCs (mean latency from stimulus artifact 5.9 ± 0.3 ms) and later components, suggesting mGluRs to be present on primary afferent terminals presynaptic to motoneurones as well as in pathways via interneurones. These results are consistent with the presence of at least two types of presynaptic mGluR that modulate release of glutamate in segmental pathways convergent onto motoneurones. These receptors appear to be activated by interstitial glutamate tonically present in the present preparations. © 1997 Elsevier Science Ltd. All rights reserved.

1. In the immature rat in vitro hemisected spinal cord preparation the dorsal root-evoked depolarizing potential recorded from an adjacent dorsal root DR-DRP had a mean peak amplitude (+/- s.e.mean, n = 27) of 2.9 +/- 0.2 mV and a mean latency to peak amplitude of 106 +/- 3 ms. The DR-DRP amplitude was maximal with a stimulus intensity of four times the threshold intensity required to activate the lowest threshold fibres. The peak amplitude and/or integral over a time-source of 0.5 s were used to assess the effects of applied drugs. 2. The DR-DRP was abolished by baclofen (mean IC50 190 +/- 46 nM, n = 7). The depressant effect of baclofen was reversed by CGP35348 (1 mM). The mean apparent Kd value calculated from dose-ratios was 16.7 +/- 6.4 microM (n = 3). 3. At a maximally effective concentration, tizanidine (1 microM) produced at the most only a 14% depression of the DR-DRP (n = 4). Clonidine (0.3 microM) had an effect similar to that of tizanidine. These depressant effects were reversed by idazoxan (1 microM). 4. The DR-DRP was potentiated by diazepam in a flumazenil (1 microM)-reversible manner. A maximal potentiation of 23.2 +/- 2.7% (n = 5) was produced by 1 microM diazepam. 5. Diazepam (1 microM) induced a mean bicuculline- (10 microM, n = 2) and flumazenil- (1 microM, n = 8) sensitive depolarization in the dorsal root of 0.25 +/- 0.03 mV (n = 8). However, diazepam failed to depolarize dorsal roots (n = 3) which had been excised from the spinal cord. 6. Comparison of the above effects with previously reported depressant effects of these drugs on the synaptic output from ventral roots suggests that actions on presynaptic inhibition, as reflected in the DR-DRP, are of subsidiary importance in explaining the muscle relaxant actions of tizanidine or diazepam.

The effects of substance P and the selective neurokinin-1 receptor antagonist (+/-)-CP-96,345 have been compared on in vitro spinal cord preparations from the rat and the gerbil. Substance P produced a concentration-dependent depolarization of motoneurons recorded from ventral roots of both species. The EC50 values (microM mean +/- S.E.M.) obtained in rat (0.95 + 1.0/-0.49) and gerbil (0.47 + 0.26/-0.17) preparations were comparable. The mean maximal depolarization (mV mean +/- S.E.M.) evoked in rat (2.07 + 0.26/-0.25) was approximately two-fold greater than that evoked in gerbil (1.21 + 0.15/-0.14) preparations. In the rat substance P had a biphasic effect (depression followed by potentiation) on the short latency probably monosynaptic reflex evoked by electrical stimulation of a dorsal root. In gerbil preparations substance P produced only potentiation of the monosynaptic reflex. The EC50 values (microM) mean +/- S.E.M.) for this potentiating action in rat (0.97 + 0.75/-0.43) and gerbil (0.46 + 3.6/-0.4) preparations were similar. This potentiation demonstrates a positive modulation of an endogenous excitatory probably glutamatergic transmission by substance P in the ventral horn of the spinal cord. The depressant phase observed in rat preparations may be related to the relative immaturity of myelination in rat ventral root fibres compared to the gerbil. The selective neurokinin-1 antagonist (+/-)-CP-96,345 was one hundred-fold less potent as an antagonist of substance P-induced depolarizations in the rat (pA2 4.69 +/- 0.18, n = 7) than in the gerbil (pA2 6.79 +/- 0.16, n = 5) spinal cord. This finding suggests that (+/-)-CP-96,345 may not act solely at the neurokinin-1 recognition site. In conclusion this study demonstrates that substance P modulates the monosynaptic reflex in the spinal cord presumably via activation of neurokinin-1 receptors.

A 10-year-old male Domestic Longhair cat with weight loss, enlarged left testicle, icterus and mild liver enlargement showed, at necropsy, two white, raised, firm, circumscribed masses in the liver and striking enlargement of the left testicle. The histological features of the liver masses were consistent with lymphangioma. Severe, diffuse, coalescing to multifocal, subacute, necrotizing orchitis of the left testicle also was diagnosed. A literature review and examination of the epidemiology of lymphangioma in small domestic animals demonstrated the rarity of this tumour in cats.

1. The shift in d.c. potential in dorsal roots (EC50 8.0 microM +/- 0.9 s.e. mean, n = 5) or depression of the C elevation of the compound action potential (EC50 3.0 microM +/- 0.3, n = 7) have been used to measure the depolarizing action of kainate on dorsal root C fibres of immature (3 to 5 day old) rats. Depolarization of motoneurones was measured from the shift in d.c. potential in ventral roots. 2. 6-Cyano-7-nitroquinoxaline,2-3,dione (CNQX) (pA2 5.78 +/- 0.06, n = 8) and 6-nitro-7-suplhamobenzo(f)quinoxaline-2,3-dione (NBQX) (pA2 5.75 +/- 0.04, n = 7) had similar potencies as antagonists of kainate at dorsal root fibres. The potency of NBQX as a kainate antagonist was similar also at motoneurones (pA2 5.72 +/- 0.07, n = 3). At motoneurones, NBQX was less potent as an antagonist of domoate (pA2 5.29 +/- 0.05) and more potent as an antagonist of S-alpha-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (pA2 6.80 +/- 0.09) than as an antagonist of kainate. 3. Application of L-glutamate, quisqualate and RS-AMPA to dorsal roots produced only short lasting depolarizations but kainate concentration-effect plots were shifted to the right in the presence of these three agonists (pA2 5.08 +/- 0.08, (n = 3), 5.59 +/- 0.04, (n = 4) and 4.46 +/- 0.04 (n = 4) respectively). Slopes of dose-ratio against concentration were significantly less than one for the latter antagonism. 4. The amplitude of depolarizations induced by L-glutamate, AMPA and quisqualate were increased up to ten fold and those induced by kainate up to two fold following treatment of dorsal roots with concanavalin A. The duration of the responses was increased also by the latter treatment. Folowing 85 s applications of glutamate, quisqualate, AMPA and kainate the mean respective times (s +/- s.e.mean (n))taken for responses to decay to half the peak amplitude were increased from 63 +/- 7 (10), 86 +/- 17 (4),95 +/- 19 (4) and 135 +/- 3 (12) to 202 +/- 49 (10), 147 +/- 7 (4), 160 +/- 13 (6) and 163 +/- 10 (10). Under similar conditions the mean decay time of y-aminobutyric acid-induced responses was 145 +/- 7 (10). This was not significantly altered by concanavalin A treatment.5. Application to dorsal roots of L-aspartate at concentrations up to 5 mm (with or without concanavalin A treatment), the selective metabotropic agonist 1S,3R-trans-1-aminocyclopentane-1,3-dicarboxylate (1 mM,) and D-serine (20 pM) in the presence or absence of N-methyl-D-aspartate (NMDA,500 pM) neither depolarized the preparations nor shifted the kainate concentration-effect plot.6. It is concluded that primary afferent C fibres possess only one type of non-NMDA receptor which is activated strongly by domoate or kainate but only weakly by AMPA. This receptor is readily desensitized by glutamate, quisqualate or AMPA and it is less readily desensitized by kainate.

1. Mature in vitro rat spinal cord preparations have been used to compare the depressant effects of 6-cyano-2,3-dihydroxy-7-nitroquinoxalinedione (CNQX) and kynurenate on transmission from low threshold myelinated primary afferents in dorsal roots. EC50 values +/- s.e.mean (number of preparations in parentheses) for depression of the monosynaptic ventral root reflex were respectively 1.0 +/- 0.3 microM (5) and 135 +/- 15 microM (3) for CNQX and kynurenate. Transmission through superior cervical ganglia was not significantly affected by concentrations of CNQX up to 100 microM or kynurenate up to 5 mM. 2. Immature in vitro rat spinal cord preparations were used to measure dose-ratios for antagonism of depolarizations induced by N-methyl-D-aspartate (NMDA), kainate or quisqualate by 4, 10 and 25 microM CNQX. In the presence of 0.75 mM Mg2+ pA2 values +/- s.e.mean were respectively 4.62 +/- 0.05 (16), 5.79 +/- 0.01 (4) and 5.59 +/- 0.05 (16) for each agonist. These values were not significantly altered in the absence of added Mg2+. The mean pA2 values for kainate were significantly higher than those for quisqualate (P less than 0.01). 3. Antagonism of NMDA-induced depolarizations was evident at 10 and 25 but not 4 microM CNQX. The antagonism of NMDA was reversed by D-serine (100 and 200 microM). 4. A similarity between the relative potencies of both CNQX and kynurenate for depression of synaptic transmission and antagonism of amino acid-induced depolarizations indicates that monosynaptic transmission from myelinated primary afferents to motoneurones is mediated by kainate and/or quisqualate sub-types of non-NMDA receptors.

Pentobarbitone (50 microM) has been shown to potentiate GABA-induced responses in rat isolated superior cervical ganglia and in dorsal or ventral root fibres of immature rat isolated spinal cords. Measurements of dose ratios for the antagonism of GABA-induced responses of dorsal root fibres by bicuculline showed that pentobarbitone (50 microM) did not significantly alter this antagonism. This result indicates that the antagonism of GABA by bucuculline at dorsal root fibres is not reversed by pentobarbitone. Ventral root responses to GABA (but not glycine) were also potentiated by pentobarbitone (+/-)-Nipecotic acid (300 microM) potentiated responses to GABA much more than those to 3-aminopropane-sulphonic acid.

The use of the procaine-blocked hemisected spinal cord preparation to identify the primary action of amino acids and their antagonists on amphibian mononeurones is described. 2 Apart from an anomalous effect of glycine, the responses of frog spinal motoneurones to amino acids were shown to be similar to those of mammalian spinal neurones. 3 In the presence of procaine, gamma-aminobutyrate (GABA), taurine and beta-alanine caused a hyperpolarizing response, measured in ventral roots, whereas L-glutamate and, to a lesser extent, glycine caused depolarization. 4 Picrotoxin and bicuculline specifically blocked ventral root responses to GABA; strychnine blocked responses to taurine and beta-alanine but not responses to L-glutamate, glycine or GABA.

The preparation of an isolated hemisected spinal cord preparation, maintained in vitro, from mature (180-300 g body weight) rats is described. Sacral and coccygeal segments (S2-Co1) gave consistent ventral root reflexes (DR-VRP) from electrical stimulation of dorsal roots. The mean latency and amplitude of the fastest component in the ventral root reflex, at 25 degrees C, were 1.6 msec +/- 0.4 SE mean and 8.2 mV +/- 0.9 SE mean, respectively (28 preparations). This component was resistant to the NMDA antagonist, 2-amino-5-phosphonopentanoate (AP5) but was depressed markedly by kynurenate. A slow component of the ventral root reflex, which was sensitive to AP5 was enhanced and spontaneous AP5-sensitive synaptic potentials sensitive to AP5 appeared in the absence of magnesium ions. The excitant amino acids L-aspartate, L-glutamate, N-methyl-D-aspartate (NMDA), kainate and quisqualate produced dose-dependent depolarizing responses in the ventral roots. The relative depolarizing potencies +/- SE mean (N) of NMDA, kainate and quisqualate, relative to L-glutamate = 1, were 96 +/- 30 (6), 234 +/- 57 (6) and 145 +/- 40 (5), respectively. These properties, apart from reduced latency of synaptic responses, are similar to those described previously for preparations from immature animals. However, it will be easier with the mature preparation to selective activate high and low threshold primary afferents.

1. Experiments were conducted in vitro on isolated spinal cords of frogs and immature rats and in vivo on cat spinal neurones. 2. The concept of two major types of excitatory amino acid receptors present in these preparations is summarized, one type (NMDA receptors) being activated specifically by N-metyl-d-aspartate (NMDA) and blocked by specific antagonists such as d(−)-2-amino-5-phosphonovalerate (APV), and a second type (non-NMDA receptors) characterized by insensitivity to specific NMDA antagonists. This second type may be comprised of two sub-types activated selectively by the agonists quisqualate and kainate. The putative transmitters l-glutamate and l-aspartate have mixed action on both NMDA and non-NMDA receptors. The major action of both transmitter candidates is considered to be on non-NMDA receptors, but the proportion of the composite responses mediated by NMDA receptors (at least for spinal neurones) appears to be greater for l-aspartate than for l-glutamate. 3. The preference of NMDA and non-NMDA receptors for a range of agonists is discussed. Some newer agonists are considered, in addition to several known agonists not previously discussed in terms of NMDA- and non-NMDA-receptor preference. Structure-activity relations of agonists are discussed. 4. The actions of some new amino acid antagonists are reported. Some of these have useful kainate and quisqualate blocking activity, in addition to their ability to block NMDA induced responses. 5. Evidence is presented suggesting that excitatory amino acid receptors are involved in both polysynaptic and monosynaptic excitation in the spiral cord, NMDA receptors mediating polysynaptic excitation and non-NMDA receptors monosynaptic excitation. 6. The unusual effect is reported of l-2-amino-4-phosphonobutyrate, which potently blocks spinal synaptic excitation in the absence of depressant action on excitatory amino acid-induced responses.

1 The nature of the catecholamine-induced contracture of chronically denervated mouse diaphragm muscle has been investigated and compared with the contractural response evoked by acetylcholine. 2 The time course of onset of catecholamine-sensitivity in denervated diaphragm muscles was similar to the development of acetylcholine sensitivity. However, catecholamine contractures were absent in tissues denervated for periods longer than 90 days whereas acetylcholine-sensitivity was still evident several months after denervation. 3 The catecholamine-induced contracture of the denervated muscle was inhibited specifically by beta-receptor blocking drugs and was unaffected by alpha-receptor blocking drugs and cholinoceptor antagonists. 4 Catecholamine-induced contractures of denervated muscles, unlike contractures to acetylcholine, were dependent upon the presence of spontaneous fibrillation and the amplitude of spontaneous fibrillation was increased by catecholamines. Fibrillation was absent in the presence of tetrodotoxin (1 muM), 2,4-dinitrophenol (10 muM), potassium cyanide (10 muM), ouabain (100 muM), in lithium chloride Ringer solution and at low temperature. Under these conditions catecholamine-induced contractures, but not those to acetylcholine, were abolished. 5 Labelled calcium was found progressively to enter denervated muscle fibres and this entry of calcium was increased by catecholamines. It is suggested that this calcium entry may represent either an increased calcium permeability of denervated muscle fibres which is increased further by catecholamines or the presence of a calcium current that occurs during the fibrillatory potentials of denervated muscle.

The effects of some central depressant drugs on amino acid-induced depolarization of motoneurons have been determined in the isolated hemisected frog spinal cord. Motoneuronsdepolarization was recorded from ventral roots and measurements were made in the presence or absence of procaine or tetrodotoxin to minimize indirect effects of both drugs and amino acids. Chlorpromazine (0.05–0.1 mM) and diazepam (0.5 mM) produced a similar differential pattern of depression of amino acid-induced depolarizations. Responses induced by L-homocysteate were markedly antagonized by these drugs, while responses to quisqualate were unaffected. L-Aspartate-induced responses were antagonized more than L-glutamate-induced responses. This pattern of antagonism resembles that previously described for Mg2+. In contrast, pentobarbital (0.1 or 0.3 mM), and the inhibitory amino acids GABA and β-alanine (0.5–1.0 mM), depressed amino acid-induced responses in a more uniform manner. The differential effects observed with chlorpromazine and diazepam provide further support for the possibility that responses to excitant amino acids structurally related to L-glutamate may have different underlying mechanisms.

In the present experiments the dorsal root-evoked dorsal root potential (DR-DRP) has been measured in vitro from a mature rat sacrococcygeal preparation. The DR-DRP is an index of presynaptic inhibition since it represents the depolarization of primary afferent terminals by γ-aminobutyric acid (GABA) released synaptically from interneurones. The present study shows that the synaptic excitation of the GABAergic interneurons contains a large component resistant to the selective N- methyl- d -aspartate (NMDA) receptor antagonists 2-amino-5-phosphonopentanoate (AP5) (100 μM) and 3((+)-2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP) 20–100 μM. This non-NMDA receptor mediated component reflected in the DR-DRP was depressed markedly by the non-selective excitatory amino acid receptor antagonists kynurenate (1–2 mM) and 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX) (10–20 μM). Because previous reports show non-cholinergic activation of Renshaw cells to be blocked by NMDA receptor antagonists the present observations suggest that pre- and postsynaptic inhibition in the spinal cord are mediated by different types of excitatory amino acid receptor.

A mature sacrococcygeal in vitro spinal preparation from the rat has been used to demonstrate effects of neutral amino acids and their antagonists. gamma-Aminobutanoate (GABA), glycine and taurine (0.5-5 mM) produced dose-dependent depression of spontaneous paroxysmal activity generated in Mg2+ -free medium. The depressant effect of GABA was antagonised selectively by picrotoxin (25-50 microM) and the depressant effects of glycine and taurine were antagonised selectively by strychnine (0.2 microM). Glycine (0.5-5 mM) had a dose-dependent depolarizing action which was present at the central ends of isolated ventral roots. gamma-Aminobutanoate and taurine, had only weak depolarizing actions on ventral root fibres. Depolarizing responses to glycine showed a marked fading. Reduction in the fading appeared to be responsible for a paradoxical potentiation of glycine-induced depolarizations, which occurred in the presence of strychnine (0.2-2 microM). Strychnine (2-10 microM), picrotoxin (10-50 microM) or bicuculline (10 microM) had little or no effect on the amplitude, duration or latency of the monosynaptic component of ventral root reflexes evoked by supramaximal stimulation of dorsal roots (DR-VRP). However all three antagonists introduced slow, NMDA receptor mediated, components to these ventral root potentials. Picrotoxin and bicuculline, but not strychnine, reversibly depressed the dorsal root potential evoked from an adjacent dorsal root (DR-DRP). The depressant actions of 2-amino-5-phosphonopentanoate (AP5), kynurenate and 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) revealed both NMDA and non-NMDA receptor mediated components in the dorsal root potential.

1. 1. The ED50 for loss of righting behaviour of cockroaches induced by kainate (43 μ mol/kg body weight) indicated the toxicity of kainate to be much greater than would have been predicted from the excitatory action of this amino acid at insect skeletal muscle fibres. N-Methyl-D-aspartate had little effect on righting behaviour (ED50 > 3500 μ mol/g body weight). 2. 2. Electrical recordings from the locust ventral nerve cord showed kainate (0.1–2 mM) to have a depolarizing action on neurones within the metathoracic ganglion. The depolarizing action of kainate was partially resistant to tetrodotoxin. 3. 3. The kainate-induced abolition of rostrally evoked potentials recorded in the abdominal connectives from the metathoracic ganglion suggests that the giant fibres are sensitive to kainate. Domoic acid was 46 times more potent than kainate. 4. 4. The lack of effect of N-methyl-D-aspartate (2 mM), dihydrokainate (2 mM), quisqualate (2 mM) and L-glutamate (20 mM) on nerve cords in the present experiments suggests that the kainate receptors in this preparation show a chemical selectivity comparable to that observed at vertebrate central neurones. The molar potencies, relative to L-glutamate, have been calculated from the present results (immobilization of cockroaches) and the sources indicated (arthropod skeletal muscle).

1. 1. Structure-activity of primary afferent depolarising action (PAD) mediated by γ-aminobutyrate (GABA) analogues suggests a difference between subsynaptic receptors located at fibre terminations within the dorsal horn and axonal receptors which are distributed throughout non-synaptic regions. 2. 2. The interaction of the bicuculline-sensitive GABA receptor (GABAA) ionophore complex with barbiturates and benzodiazepines suggests that at least three binding sites are required to explain the independent GABA-mimetic, GABA-potentiating and picrotoxin-reversing effects of such agents. 3. 3. Difficulties with explanation of the depressant effects of baclofen on spinal transmission, in terms of the bicuculline-resistant GABA (GABAB) receptor hypothesis, are mentioned. 4. 4. Glutamate-induced PAD of low threshold afferents is mediated indirectly through release of potassium. However, such terminals possess receptors (possibly autoreceptors for l -glutamate), activated by (+)2-amino-4-phosphonobutyrate, which cause depression of transmitter release. 5. 5. Primary afferent C-fibres possess receptors which are selectively activated by kainate and which mediate picrotoxin-resistant PAD. Such receptors may be involved in the presynaptic conditioning of C-fibre transmitter release. 6. 6. The peripheral terminals of vestibular primary afferents, in amphibia, possess excitatory amino acid receptors which are probably activated by the transmitter released from hair cells.

1 A series of compounds structurally related to glutamic acid has been tested on frog and rat spinal neurones. The substances were added to procaine-containing medium bathing the isolated hemiscected spinal cord of the frog, and their potencies in depolarizing motoneurones were assessed by the magnitude of the potential produced in the ventral root. The electrophoretic technique was used to administer the substances around single interneurones of the rat spinal cord and the relative potencies of the compounds as excitants assessed by the magnitude of the currents required to produce similar rates of neuronal firing. 2 Parallel structure-activity relations were observed in the two series of experiments, suggesting that the receptors for excitatory amino acids on frog and rat spinal neurones are similar. 3 Quisqualate, domoate and kainate were the strongest excitants in both animals, with potencies around two orders of magnitude higher than that of L-glutamate. 4 2,4,5-Trihydroxyphenylalanine (6-OH-DOPA) was a stronger excitant and L-3,4-dihydroxyphenylalanine (L-dopa) a weaker excotamt than L-glutamate on frog spinal motoneurones. The former compounds was also a more potent convulsant than L-glutamate on intraventricular injection into mouse brain. The lack of activity of 6-OH-DOPA on electrophoretic administration was attributed to oxidation. 5 Unlike the majority of amino acid excitants, several of the compounds shown in the present work to have moderate excitatory activity are not anionic at physiological pH. This indicates either that two negatively charged groups are not essential for interaction with a common excitatory receptor, or that more than one type of receptor is involved in the actions demonstrated.

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1. A range of compounds has been tested for excitatory amino acid agonist or antagonist activity and for effects on synaptic activity on isolated hemisected spinal cords of frogs. 2. L-Monoamino dicarboxylic acids of chain length up to 8 carbon atoms (L-alpha-aminosuberate) were all agonists. 3. Within a series of D-monoamino dicarboxylic acids, and with diamino dicarboxylic acids (mainly unresolved mixtures of diasteroisomers), there was a progression from agonist activity, for compounds of chain length equal to or shorter than glutamate, to antagonist activity, for compounds of longer chain length equal to or shorter than glutamate, to antagonist activity, for compounds of longer chain length, D-alpha-Aminosuberate (D alpha SD) was the most potent antagonist. 4. The antagonist actions of these substances showed a Mg2+--like selectivity with respect to depolarizations produced by different excitants. N-methyl-D-aspartate (NMDA) was the most susceptible agonist and quisqualate and kainate the least susceptible. Responses to other excitatory amino acids, including L-glutamate and L-aspartate, showed intermediate sensitivity to the antagonists. 5. A parallelism was observed between the relative potencies of mono- and diamino dicarboxylic acids as NMDA antagonists and their relative potencies as depressants of synaptic responses. 6. The results support the concept of different types of excitatory amino acid receptors, with NMDA and its antagonists acting predominantly on one type. These NMDA receptors are probably transmitter receptors activated by an excitatory amino acid transmitter.